AKG Slows Brain Aging by Dialing Down the mTOR Pathway

Oxidative stress is a central driver of brain aging and neurodegeneration, making antioxidant and metabolic interventions attractive therapeutic targets. Alpha-ketoglutarate (AKG), a key intermediate in the tricarboxylic acid (TCA) cycle, has previously been linked to longevity and oxidative resilience across species, but its specific mechanisms in neuronal aging were poorly understood. This study provides the first systematic mechanistic account of AKG's neuroprotective role, integrating cell biology, animal behavior, and proteomics.

In vitro, hydrogen peroxide (H₂O₂)-treated HT22 mouse hippocampal neurons served as an oxidative senescence model. AKG pretreatment dose-dependently reversed cytotoxicity, restored proliferative capacity (EdU incorporation), reduced intracellular ROS by flow cytometry, elevated SOD and GSH antioxidant activity, and lowered MDA lipid peroxidation. Critically, AKG suppressed the senescence effectors p53 and p21 at the protein level and curtailed the senescence-associated secretory phenotype (SASP), reducing pro-inflammatory cytokines including CXCL-1, TNF-α, IL-1β, and IL-6. Mitochondrial function was also restored, as shown by improved JC-1 mitochondrial membrane potential, elevated ATP production, and an increased NAD⁺/NADH ratio.

In vivo, C57BL/6 mice received D-galactose to induce accelerated brain aging, with AKG administered as a dietary supplement at multiple doses. Behavioral testing showed dose-dependent improvements in Morris water maze performance (reduced escape latency, increased platform crossings, longer target quadrant dwell time), rotarod latency to fall, and passive avoidance accuracy. Brain and plasma antioxidant markers (SOD, GSH) rose while oxidative damage markers (MDA, protein carbonyls) fell, recapitulating the in vitro findings systemically. Mitochondrial membrane potential and ATP levels in brain tissue were similarly restored.

To identify the molecular mechanism, the team employed data-independent acquisition mass spectrometry (DIA-MS) proteomics on AKG-treated versus control HT22 cells, followed by KEGG pathway and GSEA enrichment analyses. mTOR signaling emerged as the top-ranked pathway modulated by AKG. Western blotting confirmed that AKG suppressed mTOR phosphorylation and activated ULK1, the autophagy-initiating kinase downstream of mTOR inhibition. This mechanistic axis — AKG → mTOR suppression → ULK1 activation → autophagy induction — provides a coherent explanation linking AKG's metabolic role to its anti-senescence effects.

Overall, this study establishes AKG as a pleiotropic metabolic compound capable of mitigating neuronal senescence through convergent antioxidant, mitochondrial, and mTOR-regulatory mechanisms. The 1% dietary supplementation dose showed maximal neuroprotection in mice, though translation to human dosing requires further study. The work is limited by reliance on surrogate aging models and lacks direct examination of autophagic flux, but it lays a compelling foundation for AKG as a candidate intervention in Alzheimer's disease, Parkinson's disease, and general age-related cognitive decline.